Method for detection of a physiological abnormality

ABSTRACT

There is disclosed a method for the determination of a physiological abnormality in a human or animal subject, which method includes determining ion flux across the membrane of epithelial cells taken from the subject, wherein said cells are selected from the group consisting of check epithelial (buccal mucosal) cells, skin dermal epithelial cells and bladder epithelial cells. In one embodiment of the invention, the ion is a sodium ion, and the physiologically abnormality is hypertension or a predisposition towards hypertension.

TECHNICAL FIELD

The present invention relates to an assay method for detecting aphysiological abnormality such as hypertension in a human or animal, andto assays and kits for use in the method.

Hypertension is considered a major risk factor for heart disease.Untreated hypertension generally leads to irreversible damage to theheart and vasculature and undoubtedly shortens life. Cardiovascularproblems associated with hypertension have decreased greatly during thelast decade due to a combination of greater community awareness of theproblem, early diagnosis by physical examination, and efficacioustreatment of the condition once detected via the wide spectrum ofantihypertensive medications currently on the market. In some caseshypertension can also be successfully contained by a combination of dietand lifestyle changes but the condition must be recognised and treatedearly if later deleterious effects are to be avoided. Nevertheless, thefact remains that hypertension is the commonest of the diseasesaffecting the heart and blood vessels.

Studies from the National Heart Foundation's Risk Factor PrevalenceStudy (1980) indicate that one in five men and one in six women inAustralia have high blood pressure. By 50 years of age, 31% of men and28% of women in Australia are hypertensive and by age 60 years this hasincreased to 47% and 39 % respectively. At present only about half ofthese persons are aware of their condition and only about one third arebeing successfully treated.

Most people suffering from the commonest variety of high blood pressure(essential hypertension which is of unknown aetiology), develop thedisease in their thirties. However, during its early stages, it rarelyproduces symptoms. Unless a physical examination reveals that the bloodpressure is high, a person may have the disease for many years withoutknowing. In general, they may be destined to develop hypertension atsome time in the future due to their genetic predisposition. In terms ofits inheritance, essential hypertension is understood to develop from apolygenic predisposition whose expression is enhanced by environmentalmechanisms such as diet and whose outcome is fixed by inevitablestructural changes due to the elevated blood pressure itself (Williamset at., 1990a,b).

The development of reliable detection techniques for the determinationof physiological abnormalities such as the predisposition tohypertension is clearly of benefit in allowing early treatment. To datethere is no satisfactory method to identify pre-hypertensive children;except perhaps, for the long-term procedure of blood pressure tracking.Standardised measurements of a child's blood pressure every two or threeyears during growth may show a consistently elevated pressure comparedwith other children of the same age, sex and body mass index and thiswill indicate a likelihood that the child will become a hypertensiveadult. Blood pressure generally rises steadily with age and the bloodpressure defined as mild hypertensive (95 mmHg diastolic and/or 150 mmHgsystolic pressure) are rarely encountered in children. Nevertheless, thetendency to track in an upper or lower percentile rank is reasonablystrong and is a good predictor of the adult ranking (Kneisley et at.,1990).

Clearly the development of a simple diagnostic test which is capable ofdetecting both the pre-hypertensive individual and those adultsexhibiting labile blood pressure readings which make a definitediagnosis difficult, would herald a major breakthrough in early anddefinitive diagnosis and would allow for earlier treatment. Such adiagnostic test would be all the more acceptable if it had wideapplication such as in the testing of children of hypertensive parents.Furthermore, if the test is essentially non-invasive in its nature, itneed not be confined to the above situations, but could be more widereaching and applied to other "at-risk" groups in the community.

BACKGROUND ART

The most desirable form of test or predictive marker would be one whichis indicative of some basic biochemical abnormality or defect in thehypertensive individual or in those individuals who would later develophypertension, and which is likely to be related to the geneticpredisposition of an individual to develop hypertension. Certainly noform of biochemical assay presently exists to detect those who may bepredisposed to develop hypertension. Furthermore no biochemical test orassay has been developed which correlates unequivocally with allestablished hypertensive individuals. Existing biochemical assays appearto identify only certain subsets of hypertensives, i.e. only a certainproportion of hypertensive individuals display biochemical activitieswhich are significantly different from the values determined for acomparable set of normotensive individuals (Garay, 1987). In thisregard, the frequencies of Na⁺ transport abnormalities in the red bloodcells of Caucasian hypertensive subjects and the resulting change in theconcentration of Na⁺ in the red blood cell have revealed abnormalitiesin four major Na⁺ transport pathways. However, as stated above, theextent to which these individual pathways are abnormal in hypertensionis certainly not uniform, nor is the ability of these Na⁺ transportabnormalities to discriminate between hypertensive and normotensiveindividuals, definitive. The maximal rate of red blood cell Na⁺ --Li⁺countertransport activity has been reported to be increased in only 20%to 50% of subjects in a hypertensive study group while that of the (Na⁺+K⁺) pump was increased in only 5% to 15% of the hypertensive group. Anincreased uptake of Na⁺ into the red blood cell via a passive "leak"pathway was reported to be present in only 10% to 30% of hypertensiveindividuals (Garay, 1987). The above are clear examples of the failureof existing tests to clearly and totally discriminate betweenhypertensive and normotensive individuals. Furthermore, they do notaddress identification of the pre-hypertensive individual.

Existing tests developed by others rely on the measurement of Na⁺,Li⁺ orH⁺ ion fluxes (whether singularly or in various combinations) across redblood cell, leucocyte or platelet cell membranes. Measurement of theseion fluxes have been done by direct means or indirectly by followingcellular pH changes or swelling of a particular cell type. These testshave only been used in respect of adult subjects with establishedessential hypertension who have been compared with an appropriatecontrol group of normotensive subjects who are matched for age, sex andbody mass index. A complete list of these twenty or more studies aresummarised in Table 6 and the full reference to each work is cited inthe reference section. It must be pointed out that for some of thesetests up to 120 ml of blood must be sampled in order to carry out suchassays. Use of these tests in the case of pre-hypertensive subjects orwith children has to our knowledge not been reported. The use of cheekcells in studies on existing hypertensives has also to our knowledge notbeen exploited by others.

DISCLOSURE OF THE INVENTION

It is an object of this invention to provide an alternative and moresensitive method for the determination of a physiological disorder suchas essential hypertension in man. We have found that the presence of theabove disorder may be reflected in the activity of a biochemical markerpresent in epithelial cells, the marker being the flow of ions,particularly Na⁺ and H⁺, across the membrane of the epithelial cell.This marker may be used to discriminate between subjects having thedisorder and those who have not, such as has been done in an adult studygroup. Furthermore this marker may be used to discriminate betweensubjects who have yet to develop this disorder but are considered to beat the greatest risk of later developing hypertension due to thecombination of blood pressure tracking characteristics and familyhistory of hypertension, as has been done in an adolescent study group.

Accordingly, in a first aspect, the present invention provides a methodfor the determination of a physiological abnormality in a human oranimal subject, which method includes determining ion flux across themembrane of isolated single epithelial cells taken from said subject.

The epithelial cell may be a skin dermal epithelial cell, a bladderepithelial cell (eg that contained in urine), a nasal epithelial-celland the like. A particularly preferred cell is the cheek epithelial(buccal mucosal) cell.

In another aspect, the present invention provides an assay for use inthe determination method of the invention said assay comprisingdetermining the flux of a first ion across the membrane of epithelialcells under an imposed concentration gradient of a second ion.

The first and second ions may be cations. The first ion may be thesodium ion (Na⁺), the second ion (H⁺) and the epithelial cell such asthe cheek epithelial (buccal mucosal) cell.

The invention will now be more particularly described in reference tothe determination of hypertension and/or pre-disposition towardshypertension in humans.

The biochemical marker assayed for the determination of humanhypertension (or pre-hypertension) is based on the rate ofproton-dependent sodium ion (Na⁺) uptake that occurs in epithelial cellssuch as cheek epithelial cells when measured as a function of theimposed proton gradient. It is likely, although not proven, that suchactivity is due in part to the Na⁺ /H⁺ antiporter or exchanger which hasbeen argued by others to be a component activity of the Na⁺ /Li⁺exchanger which is frequently measured in red blood cells and othercells derived from the blood in relation to hypertension (see Table 6).

Thus, in yet a further aspect, the present invention provides a methodfor the determination of hypertension and/or pre-disposition towardshypertension in a human subject, said method comprising the steps of:

establishing a proton (H⁺) concentration gradient across the membrane ofepithelial cells taken from said subject, the proton concentration beinggreater inside said cells than outside said cells; and

determining the proton-dependent uptake of sodium ions (Na⁺) by saidcells when said cells are exposed to medium containing sodium ion (Na⁺).

Preferably the cells are exposed to a medium containing a finalconcentration in the range 50 micromole to 150 millimole per liter Na⁺.More preferably the cells are exposed to a medium containing a finalconcentration of about 1 millimole per liter Na⁺.

The proton-dependent Na⁺ transport may be determined directly by, forexample, determining the uptake of radioactively labelled Na⁺ (i.e. ²²Na⁺). It may also be determined indirectly by using a probe, such as afluorescent probe specific for Na⁺. Changes in proton-dependent Na⁺uptake could also be determined by measuring proton (H⁺) movementsacross the cell membrane of an epithelial cell such as a cheek cell andthe resulting pH changes inside the said cell either directly orindirectly; the latter method preferably using a fluorescent probespecific for pH.

The determination of ²² Na⁺ uptake may be performed at a number ofproton gradients ranging from about 10:1 to 100:1 (inside→outside).Differences in the net rate of proton-dependent sodium (Na⁺) transportin the cheek cell and the response of the Na⁺ uptake activity of thecheek cell to the imposed proton gradient, may form the basis fordiscriminating the hypertensive (adult) and/or pre-hypertensive(adolescent) individual, from others in the study groups, as will bedescribed.

Preferably, the epithelial cells are cheek epithelial cells, althoughother epithelial cells may be used as already indicated above. Theadvantages of using cheek cells are as follows:

1. Cheek cells are easily obtained from adults through to very youngchildren and therefore the method suffers none of the problems inherentwith blood sampling.

2. Cells can be obtained in a relatively non-invasive and entirelynon-traumatic manner. Subjects may swish small amounts of water insidetheir mouth for a short period of time. The expectorate contains the"scuffed off" cheek cells.

3. A biochemical assay can be done on each individual as sufficientcells can be obtained from one person over a period of about 3 minutesfollowing the procedure outlined above and fully detailed hereinafter.

As a result of the above points, the use of cheek cells in the method ofthe invention, would be expected to enjoy wide community acceptance inboth general surveys and specific screening programs aimed at reducingthe mortality and morbidity associated with hypertension.

In yet a further aspect, the present invention provides an assay kit foruse in the method of the invention, said kit including an indicator fordetection of ion concentration in a suitable container, and an inhibitorof (Na⁺ +K⁺)ATPase activity in a suitable container. The inhibitor maybe ouabain. The indicator may be a fluorescent probe.

The assay kit for use in the method of the invention may also include anappropriate mix of organic phthalates in a suitable container. Theorganic phthalate mixture may be used in a rapid centrifugationprocedure whereby epithelial cells could be spun out of their ²² Na⁺-containing uptake solutions into the mixture.

DETAILED DESCRIPTION OF THE ASSAY METHOD

In order to measure proton-dependent sodium transport it is necessary tosubtract the background rate of non-proton dependent Na⁺ uptake (passivediffusion) and to block the possible exit of Na⁺ from the cheek cells byinhibiting (Na⁺ +K⁺)ATPase activity (the sodium pump). It is necessaryalso to have an accurate measurement of cell number preferably by usingcell protein content from each subject, in order to compare activitiesof proton-dependent Na⁺ transport occurring in cheek cells of differentindividuals.

The transport of Na⁺ may be measured using radioactive sodium ions (²²Na⁺) after cheek cells are either acidified by incubating in pH 5.5buffer (for proton-dependent ²² Na⁺ uptake), or kept in pH 7.8 buffer(for non proton-dependent ²² Na⁺ uptake) and then added to media ofdiffering pH values to provide a range of proton gradient values.

The proton-dependent and non-proton dependent rates of ²² Na⁺ transportinto cheek epithelial cells may be measured over various time pointsfrom as short as 15 seconds to as long as 40 minutes incubation at 25°C. For the study on adult hypertensive subjects to be described, uptakeof ²² Na⁺ was determined at 30 seconds and 5 minutes, while for theadolescent study to be describe, uptake of ²² Na⁺ was determined at 2minutes and 5 minutes incubation. The uptake reaction is terminated andthe cells containing ²² Na⁺ are collected by rapid filtration using 0.45micron Millipore filters. Trapped ²² Na⁺ is counted by beta-liquidscintillation counting and the rates of ²² Na⁺ uptake are compared on amilligram cell protein basis having determined cell protein content bythe method of Lowry et al., (1951).

A full description of the methods used for the recruitment of subjectsfor each of the studies described herein, the method of collection ofthe cheek epithelial cells, the method of measuring the Na⁺ transport bythe Na⁺ /H⁺ antiporter assay, the composition of all solutions pertinentto the said assay, the source of all chemicals and the methods used forthe analysis of dam, are given hereinafter.

The present invention will now be described in reference to theaccompanying figures. It is understood that various other modificationsand/or alterations may be made without departing from the spirit of thepresent invention as it is outlined below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the time course for sodium transport activity incheek cells of a normotensive adult. The graph shows proton dependentand non-proton dependent rates at ²² Na⁺ transport in cheek cellsdetermined at indicated pH values (inside→outside) over a period of 40minutes at 25° C., using the methods fully described in Appendix 1.Amiloride at a final concentration of 1 mM was tested at a protongradient of 100:1

FIG. 2 illustrates proton dependent (total), proton dependent+ouabainand non proton dependent (+ouabain) sodium transport rates in cheekcells of normotensive and hypertensive adults (30 sec incubation). Ratesof ²² Na⁺ uptake in cheek cells at a proton gradient (inside→outside) of100:1 were determined as described below in Testing Method 2.

FIG. 3 illustrates sodium transport in cheek cells of normotensive andhypertensive adults (5 min incubation). Proton dependent (total), nonproton dependent (control) and net proton dependent (total minuscontrol) rates of ²² Na⁺ uptake in cheek cells all, with 1 mM ouabain,were determined as described in Testing Method 2.

FIG. 4 illustrates the effect of altered proton gradient on the net rateof proton dependent sodium transport in cheek cells of normotensive andhypertensive adults. Net rates of proton dependent (total minus control)²² Na⁺ transport at 5 minutes were determined at the indicated protongradient (inside→outside) values as described in Testing Method 2.Values for the regression coefficient (r) and the 95% limits ofconfidence for each group are indicated. For the normotensive adultgroup, n=19 subjects were used for each proton gradient value. For thehypertensive adult group (n) values at each proton gradient were 17(19:1); 15 (40:1); 15 (75:1) and 17 (100:1).

FIG. 5 illustrates sodium transport rates in cheek cells of normotensiveand hypertensive adults. Net rates of proton dependent a ²² Na⁺transport (total minus control) at 5 minutes incubation at a protongradient value (inside→outside) of 75:1 were determined as described inTesting Method 2.

FIG. 6 illustrates sodium transport rates in cheek cells of low BP andhigh BP tracking adolescents (2 min incubation). Proton dependent(total), non proton dependent (control) and net proton dependent (totalminus control) rates of ²² Na⁺ uptake in cheek cells at a protongradient (inside→outside) of 100:1 were determined as described inTesting Method 3.

FIG. 7 illustrates sodium transport rates in cheek cells of low BP andhigh BP tracking adolescents (5 min inculbation). Proton dependent(total), non proton dependent (control) and net proton dependent (totalminus control) rates of ²² Na⁺ uptake in cheek cells at a protongradient (inside→outside) of 100:1 were determined as described inTesting Method 3.

FIG. 8 illustrates time course for total and miloride sensitive sodiumtransport in cheek cells of low BP and high BP tracking adolescents.Proton dependent total and proton dependent amiloride sensitive rates of²² Na⁺ uptake in cheek cells were determined at a proton gradient(inside→outside) of 100:1 over a period of 10 minutes as described inTesting Method 3.

FIG. 9 illustrates the effect of altered proton gradient on the net rateof proton dependent sodium transport in cheek cells of low BP and highBP tracking adolescents. Net rates of proton dependent ²² Na⁺ transport(total minus control) at 5 minutes were determined at the indicatedproton gradient (inside→outside) values as described in Testing Method3. Values for the regression coefficient (r), and the 95% limites ofconfidence for each group are indicated.

FIG. 10 illustrates sodium transport rates in cheek cells of individuallow BP and high BP tracking adolescents, and high BP trackingadolescents with a family history of essential hypertension. Net ratesof proton dependent ²² Na⁺ transport (total minus control) at 2 minutesincubation at a proton gradient (inside→outside) of 32:1 were determinedas described in Testing Method 3.

FIG. 11 illustrates comparison of sodium transport rates in cheek cellsof low BP and high BP tracking adolescents, and normotensive andhypertensive adults. Proton dependent (total), non proton dependent(control) and net proton dependent (total minus control) rates of ²² Na⁺uptake after 5 minutes incubation at a proton gradient (inside→outside)of 100:1 in cheek cells of low and high BP tracking adolescents andnormotensive and hypertensive adults. Measurements of sodium transportwere determined as described in Testing Methods 2 and 3.

CHARACTERISATION OF HUMAN CHEEK CELL SODIUM ION (NA⁺) TRANSPORT: (NA⁺/H⁺ ANTIPORTER ASSAY)

The method of the invention in its preferred form relies on the factthat human cheek cells display significant sodium transport activitywhich by several criteria appears to be primarily due to the Na⁺ /H⁺antiporter system. Although the Na⁺ /H⁺ antiporter system is known to bepresent in many mammalian cell types (see full reference list relatingto Table 5), to our knowledge, there has been no reports in thescientific or patent literature that human cheek cells possess thisactivity. It therefore follows that the application of such a finding tothe detection of a physiological abnormality such as the identificationby biochemical means, of hypertensive and/or prehypertensive individualsis also unique.

Human cheek cells display significant sodium transport (Na⁺ uptake)activity, and, as a result of the various conditions tested in FIG. 1,this activity is characteristic of Na⁺ uptake via the Na⁺ /H⁺ antiportersystem. The data shown in FIG. 1, which is derived using the methodsfully described in Testing Method 1, shows that greatest Na⁺ uptakeoccurs when cells are acidified at a pH of 5.5 and then Na⁺ uptake isallowed to occur in pH 7.8 media. Under these conditions, this transient100:1 outwardly-directed proton (H⁺) gradient leads to significant Na⁺uptake (as measured by ²² Na⁺). Uptake of Na⁺ is greatly reduced when noproton gradient exists, e.g. pH 5.5_(inside) →5.5_(outside) and pH7.8_(inside) →7.8_(outside), or when an inwardly-directed protongradient exists, e.g. pH 7.8_(inside) →5.5_(outside). This data stronglyinfers that Na⁺ uptake is directly coupled to proton efflux and, assuch, implies that the Na⁺ /H⁺ antiporter system can be activated inhuman cheek cells by the methods described.

As will be shown hereafter, cheek cell Na⁺ transport is also a functionof the magnitude of the imposed (outwardly-directed) proton gradient,with the amount of Na⁺ uptake generally increasing when the protongradient is increased. (It is this response however which differsbetween the study groups investigated and forms part of the presentinvention). Nevertheless, this direct relationship between the magnitudeof the proton gradient and the magnitude of the Na⁺ uptake, furtherimplicates the presence of Na⁺ /H⁺ antiporter activity in human cheekcells.

Further evidence linking Na⁺ transport activity in the cheek cell to Na⁺/H⁺ antiporter activity, is provided by the results obtained with thespecific Na⁺ /H⁺ antiporter inhibitor, amiloride. As is shown in FIG. 1,addition of 1 mM amiloride to cheek cells activated to have a 100:1outwardly-directed proton gradient, reduces Na⁺ transport activity tolevels comparable to that observed in cheek cells where no protongradient exists.

Two other characteristics of cheek cell Na⁺ transport are evident fromFIG. 1. A steady-state level of Na⁺ uptake is reached after about 10minutes (probably corresponding in time to the full dissipation of theproton gradient), and an "overshoot" or decrease in the rate of the Na⁺uptake occurs after that time. Both of these observations arecharacteristic of the profile of Na⁺ /H⁺ antiporter activity obtained inother cell types (see for examples references in Table 5) and furtherimplicates the Na⁺ /H⁺ antiporter as being the predominant systeminvolved in Na⁺ transport in the human cheek cell.

The assay conditions for determining cheek cell Na⁺ transport activitywhich were routinely used in the studies to validate this invention, aremodified from existing methods for assaying Na⁺ /H⁺ antiporter activitypresent in other tissues and cell types (as indicated in Testing Method1). Our modifications are based on empirical observations using cheekcells which were made during the early course of the studies describedherein, and no attempt is made to exhaustively investigate all thecharacteristics of human cheek cell Na⁺ transport and the role of theNa⁺ /H⁺ antiporter in this transport system. Not withstanding this, thefollowing observations have been made with respect to Na⁺ transport andNa⁺ /H⁺ antiporter activities in human cheek cells, and these havehitherto not been previously reported:

Rates of Na⁺ uptake by human cheek cells are comparable to the reportedrates of Na⁺ uptake (via the Na⁺ /H⁺ antiporter system) in other celland tissue types as outlined in Table 5, even when allowance is made forthe wide variety of assay methods employed in the various studieslisted.

The final concentration of Na⁺ in all the cheek cell assays described nthis invention, is 1 mM. As such, this concentration is well below theK_(m) (Michaelis-Menton constant) for the cheek cell Na⁺ /H⁺ antiportersystem which is between 6 mM and 10 mM Na⁺ for a normotensive adultsubject.

The K_(m) for the internal proton binding site of the Na⁺ /H⁺ antiporterof cheek cells from a normotensive adult subject is about 1.7 μM (H⁺).The Hill coefficient of this site approximates 1.

Cheek cell Na⁺ /H⁺ antiporter activity is not greatly affected by the K⁺ionophore, valinomycin, indicating that cell membrane potential does notgreatly influence Na⁺ transport activity. As such, this is in agreementwith the findings reported in the scientific literature for Na⁺ /H⁺antiporter activity in other cell and tissue types.

The extent of Na⁺ transport in cheek cells having an outwardly-directedproton gradient of 100:1, is many fold higher in the presence of theionophores monensin (Na⁺ :H⁺) or nigericin (K⁺ : H⁺). Such findings alsovalidate the existence of Na⁺ /H⁺ antiporter activity in human cheekcells.

The transport of Na⁺ into cheek cells following the activation of theNa⁺ /H⁺ antiporter system by an outwardly-directed proton gradient,represents the net transport of the Na⁺ ions, which is, in turn, the sumof the rate of Na⁺ uptake and the rate of Na⁺ efflux. Efflux of Na⁺ fromthe cheek cell occurs simultaneously to, and is dependent on, theactivation of the Na⁺ /H⁺ antiporter system and the resulting increasein cheek cell ²² Na⁺ content. However, up to a time of 10 minutes, Na⁺influx exceeds Na⁺ efflux. The rate of Na⁺ efflux is also time-dependentand hence dependent on the extent of uptake of Na⁺ into the cheek cellwhich has occurred via the Na⁺ /H⁺ antiporter. The "overshoot"phenomenon mentioned previously, may represent a situation of greaterNa⁺ efflux than influx after complete dissipation of the proton gradientand subsequent deactivation of the Na⁺ /H⁺ antiporter activity.

The contribution of other known Na⁺ transport systems to the nettransport of Na⁺ in human cheek cells is not great. Efflux of(transported) ²² Na⁺ out of the cheek cell via the Na⁺ +K⁺ -ATPase(sodium pump), does affect the net rate of Na⁺ influx to a small butsignificant extent (see FIG. 2). However, to entirely eliminate anycontribution by this Na⁺ transport pathway to net cheek cell Na⁺transport activity, 1 mM ouabain, a specific inhibitor of the sodiumpump, is routinely included in all those assays concerned with thestudies that validate this invention (as indicated in Testing Methods2-3). On the basis of inhibitor studies using bumetanide and frusemide,the contribution of the Na⁺ /K⁺ /2Cl⁻ cotransport system to the overallNa⁺ transport activity of human cheek cells, is insignificant.

Human cheek cells also contain a significant level of Na⁺ /Li⁺ antiportactivity in which Na⁺ uptake into cells is coupled to an outwardlydirected Li⁺ gradient.

In conclusion, the Na⁺ transport activity of human cheek cells, which isused to validate this invention, has only partly been characterised atthe biochemical level. However it is now firmly established that the Na⁺/H⁺ antiporter system is largely responsible for the observed Na⁺transport. All the abovementioned characteristics of Na⁺ transport inhuman cheek cells therefore represent new and unique findings notpreviously published in the scientific or patent literature.

The discovery of the potential for human cheek cells to transport Na⁺ ina predictable manner, forms the basis for the conclusion thathypertensive and prehypertensive individuals can be identified using abiochemical marker. These specific details of the invention will now bedescribed.

Adult Cheek Cell Study

Subjects used in this study were recruited from adult volunteersidentified following several blood pressure screening programs. None ofthe control or hypertensive subjects were receiving anti-hypertensivemedication at the time of the blood pressure screening or sampling ofcheek cells. All measurements were done using a Dinamap (automatic bloodpressure recording instrument). Those first identified as being mildlyhypertensive (diastolic blood pressure ≧95 mm Hg), were subsequentlyre-measured over a period of several days to confrim the reading.Systolic and diastolic blood pressures, age and gender of the two groupsused in this example are shown in Table 1. Systolic and diastolic bloodpressures were significantly different between the two groups, while thegroups were well matched for age and gender, although the hypertensivegroup exhibited a slightly greater body mass index (BMI).

                                      TABLE 1    __________________________________________________________________________    CHARACTERISTICS OF    NORMOTENSIVE AND HYPERTENSIVE SUBJECTS USED IN THE    CHEEK CELL ASSAY FOR HUMAN HYPERTENSION                BLOOD PRESSURE                SYSTOLIC                       DIASTOLIC                              AGE       BMI             (n)                (mm Hg)                       (mm Hg)                              (years)                                  M/F                                     % F                                        (Kg/m.sup.2)    __________________________________________________________________________    Normotensive             19  129 ± 2.sup.1                        73 ± 2                              47 ± 2                                  13/6                                     31.5                                        23.4 ± 0.8    Hypertensive             17 166 ± 5                       101 ± 2                              49 ± 2                                  12/5                                     29.4                                        25.9 ± 1.0              15.sup.2                168 ± 5                       102 ± 2                              50 ± 2                                  10/5                                     33.3                                        26.0 ± 1.0    Significance                P < 0.0001                       P < 0.0001                              N.S.                                  -- -- P < 0.05    __________________________________________________________________________     .sup.1 Data is presented as the mean ± SEM for the indicated number of     subjects (n) in each group. Differences between means were determined by     Student's unpaired ttest significant.     .sup.2 For proton gradients performed at 19:1, 40:1 and 75:1.

Cheek cells were isolated from all subjects in the morning and assayedin the afternoon of the same day as collection. Proton-dependent sodiumtransport was determined using triplicate assays of both the totalsodium uptake rate and the non proton dependent rate to determine theproton-dependent sodium uptake rate, as described in the appropriatemethods of Testing Method 2. The variability of the measurements acrossall assay conditions for any one individual averaged 6.2% (interassayvariation), while for assays on the same subject(s) performed ondifferent days, the variability averaged 10.6% (intra-individualvariation).

FIG. 2 compares Na⁺ transport in cheek cells of normotensive andhypertensive adult study groups after the Na⁺ transport assay has beenallowed to proceed for 30 seconds. As indicated in the legend to thisfigure, the total rate of Na⁺ uptake is that observed when anoutwardly-directed proton gradient of 100:1 is applied to cheek cells.Inclusion of 1 mM ouabain, a specific inhibitor of (Na⁺ +K⁺)-ATPaseactivity, results in a small increase in Na⁺ uptake in both normotensiveand hypertensive adult subjects. As discussed in the previous section,this result suggests that the contribution of the sodium pump to Na⁺efflux, is minimal. Ouabain (at 1 mM) is added to all assays in whichNa⁺ transport activity of adults and adolescent study groups isinvestigated to completely negate any contribution from the sodium pump.It is also clearly apparent from FIG. 2, that the total and total plusouabain rates of Na⁺ uptake in cheek cells of the adult hypertensivestudy group is only about 50% of that evident in cheek cells from thenormotensive study group. Furthermore, this difference in Na⁺ uptakerates for the above conditions is highly significant between the twostudy groups. For hypertensive adult subjects, the control rate of Na⁺uptake in the presence of ouabain, (which represents the rate of"passive" Na⁺ uptake into cheek cells in which Na⁺ /H⁺ antiporteractivity has not been activated by a proton gradient), is also muchlower than that for the normotensive study group, but statisticalsignificance is not achieved.

FIG. 3 compares Na⁺ transport in cheek cells of normotensive andhypertensive adult study groups after the Na⁺ transport assay has beenallowed to proceed for 5 minutes. It is clear that the total and the netrates of Na⁺ uptake for the hypertensive study group are only about 50%of that evident for the normotensive study group. Furthermore, thisdifference in the (proton-dependent) Na⁺ uptake rates is highlysignificant between the two study groups. The control (non-protondependent rate) of Na⁺ uptake is not significantly different between thestudy groups.

The difference in cheek cell Na⁺ transport activity between normotensiveand hypertensive study groups is clearly apparent only under conditionswhich would lead to activation of the Na⁺ /H⁺ antiporter system, i.e.the presence of an outwardly-directed proton gradient. This factindicates that intrinsic differences in the activity of the Na⁺ /H⁺antiporter system and/or its activation by a proton gradient, mayunderlie the dramatic difference seen in the Na⁺ transport activitybetween the normotensive and the hypertensive study groups. This hasbeen further investigated in cheek cells obtained from the respectiveadult study groups, by comparing the response of the Na⁺ /H⁺ antiportersystem (and hence Na⁺ uptake), to outwardly-directed proton gradientswhich differ in their magnitude.

The response of cheek cell proton-dependent sodium transport to protongradients varying in magnitude from 19:1 to 100:1 (inside→outside) inthe normotensive and hypertensive study groups is shown in FIG. 4. Aclear distinction is apparent between the two groups. Not only is therate of proton-dependent sodium transport in the hypertensive subjectsmarkedly reduced at all proton gradient values as compared to thenormotensive group, but for the hypertensives, the rate ofproton-dependent sodium transport is unaffected by a change in protongradient values from 19:1 to 75:1. Only at a proton gradient value of100:1 does the rate of proton-dependent sodium transport of thehypertensive group increase above the value exhibited for protongradient values of 19:1, 40:1 and 75:1. The difference in the ratebetween the two groups is extremely significant for all but the 19:1value of the proton gradient.

The rate of proton-dependent sodium transport in cheek cells as a resultof imposing a proton gradient of 75:1 (inside→outside) for 15 individualhypertensives and 19 normotensive subjects, is shown in FIG. 5. A protongradient of 75:1 refers to the ratio between the hydrogen ionconcentration in the cell loading buffer and the assay (uptake buffer)respectively, after taking into account the dilution effects arising asa result of the manner in which the transport assay is performed. For a75:1 proton gradient to be achieved, the cell loading buffer has a valueof pH 5.5 and the assay uptake buffer a value of pH 7.65!. Cheek cellsfrom the 15 hypertensive subjects display a markedly reduced mean rateof proton-dependent sodium transport (mean±SEM; 0.41±0.1 nmol Na⁺.mgprotein.5 min) which is 34% of the rate for the 19 normotensive subjects(1.20±0.16 nmol Na⁺.mg protein.5 min), and this difference is highlysignificant (P=0.0004; Student's unpaired t-test). Some overlap betweenthe values for hypertensive and normotensive subjects is apparent inthis data (approximately 6% as defined in Table 6). However as discussedin relation to Table 6, this degree of overlap is less than thatobtained in any previous study where the rates of cation transport inother cell types have been measured in relation to hypertension.

The response of cheek cell proton-dependent sodium transport to a changein the value of the proton gradient from 19:1 to 75:1, is shown in Table2. These values represent the slope of the line between theabovementioned proton gradient values as depicted in FIG. 4. As can beseen from this table, the slope of the line between the normotensive andhypertensive subjects differs by a factor of 24 fold at a significancelevel of P=0.0028; i.e. the mean increase in proton-dependent ²² Na⁺transport is 24 times greater in the normotensive subjects as comparedto the hypertensive subjects. For the above parameter ofproton-dependent sodium transport, the extent of overlap between thevalues for the individual normotensive and hypertensive subjects is11.5% as defined in Table 6.

                  TABLE 2    ______________________________________    RESPONSE OF CHEEK CELL    PROTON-DEPENDENT SODIUM TRANSPORT TO    A CHANGE IN THE PROTON GRADIENT IN NORMO-    TENSIVE AND HYPERTENSIVE ADULT SUBJECTS    ______________________________________    Normotensive             n = 19  0.48 ± 0.1.sup.1                               nmol .sup.22 Na · mg protein                               · 5                               min (for a change in the pro-                               ton gradient from 19:1 to                               75:1)    Hypertensive             n = 15  0.02 ± 0.1    Significance     P = 0.0028    ______________________________________     .sup.1 Data is presented as the mean ± SEM for the indicated number of     subjects (n) in each group. The significance of the difference between th     mean was determined by Student's unpaired ttest.

The present invention has significant potential in the determination ofhuman hypertension. Marked differences in the rate of proton-dependent²² Na⁺ transport into cheek cells are apparent between age andsex-matched hypertensives and normotensive subjects under a wide rangeof assay conditions. However when the assay is performed at a protongradient value of 75:1 and measured over a period of 5 minutes, thedifferences between normotensive and hypertensive subjects is mostpronounced, (mean value of proton-dependent ²² Na⁺ uptake in thehypertensive group is only 33% of that evident in normotensive group);of highest statistical significance, (P value of 0.0004 between the twogroups, i.e. 1 chance in 2,500 that the result occurred by chancealone); and capable of the greatest discrimination between the twogroups (i.e. where least overlap occurs; average 6% as defined in Table6. The increase in the rate and amount of proton-dependent ²² Na⁺ uptakewhen the proton gradient is increased from a value of 19:1 to 75:1 isalso a useful parameter to discriminate between hypertensive andnormotensive subjects.

Adolescent cheek cell study

The preceding sections have clearly demonstrated that:

1. Human cheek cells have the ability to transport Na⁺ primarily viaproton gradient activation of an associated Na⁺ /H⁺ antiporter system;

2. Them is a statistically significant reduction in the magnitude of Na⁺transport activity in cheek cells from adult hypertensive subjectscompared to adult normotensive subjects;

3. The difference in Na⁺ transport activity between these adult studygroups appears related to the proton-gradient dependent cheek cell Na⁺/H⁺ antiporter system.

In order to extend these findings and test one of the claims of thisinvention, i.e. that the biochemical parameter under study viz-a-viz Na⁺transport in cheek cells, can be used to identify pre-hypertensiveindividuals, a study using adolescents is described.

Full details of the methods used in the Adolescent cheek cell study aredescribed in Testing Method 3. Based on the previously reported findingthat the tendency during childhood for blood pressure to track in anupper or lower percentile is reasonably strong, and that it is a goodpredictor of the adult ranking, adolescents who consistently tracked inan upper and lower percentile rank, were identified from blood pressurereadings taken over a period of at least four years. Details of thoseadolescents who were finally selected for this study are described inTable 3. The low blood pressure (BP) tracking group of n=24, represents4.8% of the total group of n=504 adolescents who were screened. Subjectswithin this group consistently exhibited the lowest systolic BP readingfrom the commencement of the study in 1986 to the time at which cheekcell Na⁺ transport activity was determined. In contrast, the high BPtracking group, representing 5.7% of the total group screened,consistently exhibited the highest (systolic) BP readings of all thesubjects screened. (Statistical procedures used to determine the BPranking status of these two groups of adolescents are fully described inTesting Method 3). It can be clearly seen from the data in Table 3, thatthe difference in the systolic BP between the two adolescent groupsremained significantly different (P<0.0001) over the four-year period ofthe tracking study. No significant difference in the age or BMI of thetwo groups is evident at the time the cheek cells were actually sampled.However, of particular importance is the extent by which the twoadolescent groups differ in their incidence of a definitive familyhistory of hypertension. In the high BP tracking group, 7 of the 29subjects (24%) had a definitive family history of hypertension with oneor both of the parents being treated with a recognised anti-hypertensivemedication. This contrasts with the low BP tracking group where atransient secondary hypertension (eclampsia) was evident for the motherof one of the subjects. A higher proportion of males (69%) is present inthe high BP tracking group in comparison to the low BP tracking group,in which 50% were male.

                                      TABLE 3    __________________________________________________________________________    COMPARISON OF LOW BP TRACKING    AND HIGH BP TRACKING SUBJECTS USED IN THE    CHEEK CELL STUDY OF SODIUM TRANSPORT IN ADOLESCENTS                  LOW BP HIGH BP     (H-L)    1990-1991     TRACKING                         TRACKING                                P    Δ    __________________________________________________________________________    (n)           24 (4.8%)                         29 (5.7%)                                --    M/F           12/12  20/9    AGE           16.5 ± 0.13                         16.6 ± 0.14                                NS   0    BMI (Kg/m.sup.2)                  20.8 ± 0.8                         22.9 ± 0.7                                NS   2.1    SYSTOLIC BP (1990-91)                  108 ± 1.1                         128 ± 1.3                                <0.0001                                     20 mm Hg    DIASTOLIC BP (1990-91)                   58 ± 1.2                          63 ± 1.8                                <0.008                                      5 mm Hg    FAMILY HISTORY                  1 (eclampsia)                         7 (24%)    1st generation    SYSTOLIC BP (1986-87)                  100 ± 0.7                         123 ± 0.8                                <0.0001                                     23 mm Hg    SYSTOLIC BP (1989-90)                  105 ± 0.9                         129 ± 1.0                                <0.0001                                     24 mm Hg    __________________________________________________________________________     (NS) Not significant

It must be emphasised that these groups are not directly comparable tothe normotensive and hypertensive adult study groups referred to in theprevious section. For example, none of the high BP tracking adolescentsrecorded BP readings which impinged on values characteristic of mildlyadult hypertensive subjects. However, each group displayed consistentlydifferent BP tracking behaviour over a period of 4 years up to the timeof cheek cell sampling. As such, it would be expected that a far greaterproportion of those subjects within the high BP tracking group may laterdevelop hypertension compared to the low BP tracking group. Thisprediction is further reinforced by the observation that 7 of the highBP tracking adolescents had a definitive family history of hypertension.The higher proportion of males and the slightly higher BMI of subjectswithin the high BP tracking group, (both of which are characteristics ofadult essential hypertension), is probably only a co-incidental findingin this study.

Cheek cells from adolescent subjects from each BP tracking group wereassayed for Na⁺ transport activity (²² Na⁺ uptake) on the day ofisolation using the methods fully described in Testing Method 3. FIG. 6shows the total, control and net rates of Na⁺ transport activityfollowing a 2 minute assay time course for cheek cells from subjectswithin the two adolescent study groups. Both proton-dependent (total andnet) and non-proton dependent (control) rates of Na⁺ transport, aresignificantly reduced in cheek cells from subjects in the high BPtracking group. The extent of this reduction in cheek cell Na⁺ transportactivity (about 50% of the rate apparent for the low BP tracking group),equates with the extent of the difference evident between adultnormotensive and hypertensive study groups (described in the previoussection).

FIG. 7 shows the total, control and net rates of Na⁺ transport activityfollowing a 5 minute assay time course for cheek cells from subjectswithin the two adolescent study groups. As is the case for the 2 minutetime point, both proton-dependent (total and net) and non-protondependent (control) rams of Na⁺ transport, are significantly reduced incheek cells from subjects in the high BP tracking group. Again thisequates with the extent of difference evident in the adult cheek cellstudy. The only distinction between the adult and adolescent studiesthus far, is the difference in the rate of Na⁺ transport activity in thecontrol assay situation where the absence of a proton gradient would beexpected not to activate Na⁺ /H⁺ antiporter activity. In the adolescentstudy this difference does reach statistical significance. However asimilar trend of lower (control) Na⁺ transport activity in cheek cellsfrom subjects in the adult hypertensive study group, is clearlyapparent.

The effect of amiloride, a specific inhibitor of Na⁺ /H⁺ antiporteractivity, has also been tested in the adolescent study. The rationalefor including this additional step in the assay protocol is to determinewhether the amiloride sensitive Na⁺ transport rate, which representsthat component of Na⁺ uptake dependent on the activity of the Na⁺ /H⁺antiporter, differs between the two adolescent study groups. FIG. 8shows a time course for the total and the amiloride-sensitive rates ofNa⁺ transport in cheek cells from subjects in the low and high BPtracking groups. Both total and amiloride sensitive Na⁺ transportactivity in cheek cells from subjects in the high BP tracking group aresignificantly reduced in comparison to the low BP tracking group at allfour time points examined.

This result further indicates that some property of the cheek cell Na⁺/H⁺ antiporter activity and/or its activation by a proton gradient,underlies the phenomenon observed in these studies. As was done in theadult study, the activation of the Na⁺ /H⁺ antiporter system by a protongradient has also been investigated in cheek cells obtained from therespective adolescent study groups. This was accomplished by comparingthe response of the Na⁺ /H⁺ antiporter system (and hence Na⁺ uptake), tooutwardly-directed proton gradients which differ in their magnitude.

The response of cheek cell proton-dependent sodium transport to protongradients varying in magnitude from 10:1 to 100:1 (inside→outside) inlow BP and high BP tracking adolescents is shown in FIG. 9. The rates ofproton-dependent sodium transport in the high BP tracking adolescentgroup are markedly reduced at all proton gradient values as compared tothe other adolescent study group. Furthermore, for the high BP trackinggroups, the slope for the proton gradient activation of cheek cell Na⁺transport is markedly reduced over that obtained for the low BP trackinggroup.

The response of the cheek cell proton dependent sodium transport to achange in the magnitude of the proton gradient from 10:1 to 100:1, isshown in Table 4. These values represent the slope of the line betweenthe abovementioned proton gradient values as depicted in FIG. 9. As canbe seen from this table, the slope of the line between low and high BPtracking adolescent groups differs by a factor of 2.5 fold at asignificance level of P=0.002; i.e. the mean increase inproton-dependent Na⁺ transport is 2.5 times less in the high BP trackingadolescents as compared to the low BP tracking adolescents. However, themagnitude of this difference in the slope of the proton-dependent Na⁺transport between the adolescent study groups is nowhere near the 24fold difference evident in the adult study. The fact that the magnitudeof this difference in the adolescent study was "dampened" as compared tothe adult study, may be a reflection of the different nature andcomposition of the two study groups in question.

FIG. 10 shows sodium transport rates (at a proton gradient value of32:1) in cheek cells of individual low BP and high BP trackingadolescents, and high BP tracking adolescents with a family history ofessential hypertension. Cheek cells from the high BP tracking groupdisplay a markedly reduced mean rate, (mean±SEM; 0.73±0.08 nmol Na⁺.mgprotein.5 min) of proton-dependent Na⁺ transport which is 57% of therate for the low BP tracking adolescents; (1.28±0.17 nmol Na⁺.mgprotein.5 min), and this difference is highly significant (P=0.0032;Student's unpaired t-test). The mean Na⁺ transport rate for those 7subjects in the high BP tracking groups (0.46±0.07 nmol Na⁺.mg protein.5min) is 63% when compared to the mean rate for the total high BPtracking adolescents, and 36%; (P=0.016) when compared to the mean ratefor the low BP tracking adolescents.

                  TABLE 4    ______________________________________    RESPONSE OF CHEEK CELL PROTON-DEPENDENT    SODIUM TRANSPORT TO A CHANGE    IN THE PROTON GRADIENT IN LOW BP TRACKING    AND HIGH BP TRACKING ADOLESCENT SUBJECTS    ______________________________________    Low tracking             n = 24  0.91 ± 0.15.sup.1                                nmol .sup.22 Na · mg protein                                · 5                                min (for a change in the                                proton gradient from 10:1                                to 100:1)    High tracking             n = 29  0.36 ± 0.09    Significance     P = 0.002    ______________________________________     .sup.1 Data is presented as the mean ± SEM for the indicated number of     subjects (n) in each group. The significance of the difference between th     mean was determined by Student's unpaired ttest.

The Na⁺ transport rates of high BP tracking adolescents with a familyhistory of hypertension are all below the mean value for Na⁺ transportfor the total high BP tracking group, and overlap with only 3 of the 24subjects in the low BP tracking group.

It is therefore clearly apparent that those adolescents who display botha high BP tracking profile together with a family history ofhypertension, also have the characteristic of a significantly anddistinctly low rate of protons-dependent Na⁺ transport activity in theircheek cells, and indeed can be distinguished by this lattercharacteristic. In this regard, the biochemical parameter under study inthis embodiment, i.e. cheek cell Na⁺ transport activity, displayssimilar characteristics in this group of adolescents (who may beconsidered at greatest risk of later developing hypertension) whencompared to the group of hypertensive adults described in the previoussection.

Finally, a comparison is made between the sodium transport rates ofcheek cells from the study groups within the adolescent and the adultstudies. FIG. 11 shows the total, control and net rates of Na⁺ transportin the adolescent and adult studies when examined after a 5 minute assaytime course and a 100:1 outwardly-directed proton gradient. The extentof the difference between low BP and high BP tracking adolescents on theone hand, and between normotensive and hypertensive adults on the other,is striking for all three assay variations examined. Despite the factthat there was a six-month interval between these studies, with theadolescent study following after the adult study, the overall rates ofcheek cell Na⁺ transport activity are nevertheless very similar.

Discussion

A comparison of previous studies of cation transport in red blood cells,leucocytes and platelets of hypertensive and normotensive subjects withthe present cheek cell assay for hypertension, is shown in Table 6.Twenty three other studies, most of which have used red blood cell Li⁺--Na⁺ countertransport activity to discriminate between hypertensive andnormotensive subjects, are listed in this Table. For ease of comparisonof these data with that of our own, some computational parameters havebeen calculated to allow both the extent of the difference between thenormotensive and the hypertensive rates of cation transport (Hx/Nx) andthe extent of overlap between the two groups, to be more readilycompared; (these terms are defined in the legend to Table 6).

It is immediately apparent from our data on the adult hypertensive studythat unlike the situation in all the other cited studies, the rate ofproton-dependent sodium uptake is lower in hypertensive subjects whencheek cells are used for measurement. This contrasts with the sixstudies in which Na⁺ /H⁺ antiporter activity has been measured in redblood cells, leucocytes and platelets. Li⁺ --Na⁺ countertransportactivity in red blood cells, which is most often measured as the rate ofsodium-dependent lithium efflux, is more difficult to relate to the Na⁺/H⁺ antiporter of proton-dependent sodium transport. Nevertheless, inthe studies shown, Li⁺ --Na⁺ countertransport activity in red bloodcells of hypertensive subjects is between 104% and 371% the rateobserved with normotensive subjects and the significance of thedifference between the two groups ranges from N.S. to P<0.001.

This same situation of decreased sodium transport activity is alsoapparent in the adolescent cheek cell study. For the adolescent studyhowever, the extent of overlap has not been included or compared to theother studies shown in Table 6 for the previously stated reasons thatthe adolescent study groups are not directly comparable to the studiesusing adult hypertensive and normotensive subjects. FIG. 11 showing acomparison of cheek cell sodium transport activity between the adult andadolescent studies confirms the similarity of the two studies reportedin this invention. When these combined results are viewed against thestudies of others shown in Table 6, it is clear that the parametermeasured in the cheek cell assay is behaving in a manner opposite tothat reported by others (using other cell types) in relation tohypertension (and also the prehypertension situation).

The converse finding of a lower cation transport activity in the cheekcells of our hypertensive group, (and in the BP high tracking adolescentgroup with a family history of hypertension; the "prehypertensivegroup") is at present difficult to explain in the light of the findingsfrom the cited studies. However, in this respect we have noted thatcheek cells exhibit certain properties which differ significantly fromthose found in the other types of cells cited in Table 6. One example isthe apparent inability of cheek cells to display significant osmoticbehaviour as is the case for human red blood cells. This would suggestthat a basic difference may exist in cell membrane permeability to ionsand solutes in cheek cells as opposed to many other mammalian celltypes.

It is therefore possible that some property unique to epithelial cellsmay account for our particular observation in relation to thehypertensive and normotensive groups as well as in the adolescent study.One likely explanation of our result is that the cheek cell plasmamembrane of the adult hypertensive and the "prehypertensive" group, hasa far greater permeability to protons (H⁺) than is the case for theother two comparable study groups. This would imply that with theimposition of a proton gradient (inside→outside), less protons would beavailable to couple with the cell membrane located Na⁺ /H⁺ antiporterdue to increased loss of cytosolic protons through the cell membrane;hence the finding of a lower rate of proton-dependent sodium transportin these (hypertensive/prehypertensive) groups.

An increase in cheek cell membrane permeability to protons in thehypertensive and prehypertensive groups is borne out experimentally byexamining the response of cheek cell sodium transport activity to protongradients differing in their magnitude. It is clear from the data inFIGS. 4 and 9, and Tables 2 and 4, that the slope (or response) ofsodium transport to an increasing proton gradient is significantly lessin both the above study groups across the range of proton gradientvalues examined. This would indicate that an equivalent number ofprotons is either not present (via rapid dissipation due to increasedmembrane permeability), and/or that the extent of coupling of protonefflux to sodium uptake differs in the hypertensive (andprehypertensive) groups compared to their respective control groups.

Besides a difference in membrane proton permeability and/or protoncoupling to the cheek cell Na⁺ /H⁺ antiporter, yet another explanationfor over result may lie in an enhanced permeability of the cell membraneto sodium ions, particularly in regard to the rate of efflux of Na⁺which has entered the cheek cell during the proton activation of the Na⁺/H⁺ antiporter. We have observed that efflux of ²² Na⁺ from cheek cellsis a rapid event and that Na⁺ efflux is probably occurring during theNa⁺ uptake process, albeit at a rate less than the rate of Na⁺ uptakeduring the fast 10 minutes of the assay incubation when measured at 25°C. A greater rate of efflux of transported Na⁺ ions in the cheek cellsof the hypertensive and prehypertensive groups compared to theirrespective control groups, could account for our experimental findings.

An important difference between the cheek cell assay method describedherein and those studies of others referred to in Table 6, is the finalconcentrations of sodium ion (Na⁺) used in the respective assays. Incontrast to the studies listed in Table 6, all of which used a finalconcentration of about 150 mM Na⁺ and did not rely on a radioisotopictechnique for assay, the present invention uses a final concentration of1 mM Na⁺ containing 4.3 to 6.4 μCi (159-237 kBq) ²² Na⁺ per μmole Na⁺.As has been previously mentioned in the section dealing with thecharacterisation of cheek cell Na⁺ transport and Na⁺ /H⁺ antiporteractivity, a final concentration of 1 mM Na⁺ is well below the Km valueof Na⁺ for the cheek cell Na⁺ /H⁺ antiporter. As such, in our assay thistransport system would be operating with Na.sup. + transport rates manytimes less than the maximum rate achievable if Michaelis-Menton kineticswere operative and the substrate (Na⁺) was saturating. This is in directcontrast to the situation in the other studies listed in Table 6 inwhich the concentration of Na⁺ in those assays would be saturating andthe Na⁺ /H⁺ antiporter would be operating at its maximum rate.

As a consequence of this important difference in the manner in which thesodium transport assays were carried out between this and the othercited studies, it would be expected that the respective assays wouldexhibit a differential sensitivity to those parameters which may affectthe rate of sodium ion transport by the Na⁺ /H⁺ antiporter. Thus, undersaturating (high Na⁺ assay concentration) conditions, differences in theabsolute mount of Na⁺ /H⁺ antiporter catalytic unit(s) present, may bemore readily observable than would be the situation when substrate islimiting as is the case in the presently described cheek cell assay. Onthe other hand however, at low substrate (Na⁺) concentrations where theactivity of the Na⁺ /H⁺ antiporter is well below its maximum rate, theinfluence of other factors, such as proton permeability may be moreapparent. This may come about due to the fact that theoutwardly-directed proton gradient is not being rapidly dissipated viathe Na⁺ /H⁺ antiporter because of the latter's relatively low activity.Dissipation of protons (H⁺) could then occur via other mechanisms whichmay involve H⁺ permeation through the cell membrane, and this rate of H⁺dissipation may represent an intrinsic and heritable characteristic ofhuman essential hypertension. As such, the cheek cell assay methoddescribed herein may be inherently more sensitive to those biochemicalfactors which are perturbed in hypertension in comparison to the methodsof others described in Table 6 which neither use cheek cells nor theassay techniques described in this invention.

In comparison to the studies cited in Table 6, it is clearly evidentthat for the adult cheek cell study, the difference in cation transportactivity between normotensive and hypertensive subjects (irrespective ofwhich group has the greater rate), exhibits the following:

(i) the greatest magnitude; Nx/Hx=2400%

(ii) the greatest statistical significance; P<0.0004

(iii) the least overlap; 6%.

With reference to (iii): Overlap has been arbitrarily defined in Table 6and data to calculate this parameter is only available in a limitednumber of studies. Attention is drawn to the Canessa study (Canessa etat., 1980) in which an overlap value of 0% was first reported. In theirfollow-up study (Semplicini et al., 1989) in which identical methods tothe Canessa et al., (1980) study were employed, the overlap valueaveraged 26%.

The studies outlined in Table 6, particularly those in which Na⁺ /H⁺(rather than Li⁺ --Na⁺) countertransport activities have beendetermined, do not show as great a difference between the two groups(141% to 195%) as found using the cheek cell assay method (293% to2400%;Nx/Hx). This finding may be due to the fact that this presentstudy was unique in that a range of proton gradient values wereinvestigated with respect to proton-dependent sodium transport and thatthis present assay concentration of Na⁺ was 1 mM.

Conclusions

It will be seen that the present invention is significant in that itachieves the following:

1) It allows the activity of a biochemical marker to be correlated withphysiological disorders such as human hypertension in cheek cells whichcan be obtained in a relatively non-invasive manner.

2) It allows the potential for screening those people in the communitywho may be genetically predisposed to develop hypertension but are stillin the prehypertensive state. Included in this category would be thoseadolescents who have high BP tracking characteristics and a familyhistory of hypertension.

3) It is noted that this latter property is particularly significant inthat for those who are identified as being in the prehypertensive stage,early measures could be taken to ameliorate later development ofhypertension, or, at the very least, to minimise the extent of thehypertensive disorder.

This invention provides an assay which can be used to detect differencesin the activity of a biochemical marker present in epithelial cellswhich can, with a minimum of overlap and with high statisticalprobability, discriminate between adult hypertensive and normotensivesubjects, and between adolescents who have a family history ofhypertension and high blood pressure tracking characteristics and thoseadolescents who do not. For the adult, the invention achieves a level ofdiscrimination which is greater than any existing test done with cellsderived from the blood of hypertensive subjects. For the adolescent, nosuch biochemical test has been described in the scientific literature towhich the present invention can be compared.

Finally, it is clearly apparent from the examples given, that we havedeveloped a technique which is far less invasive, and has greaterdiscriminatory ability than all other tests so far described in theprior art.

Modifications to the Assay Method

The transport of sodium ions (Na⁺) could also be measured using afluorescent probe sensitive to small changes in Na⁺ concentration in thecytoplasm of cheek cells resulting from Na⁺ /H⁺ antiporter activity.Such a probe (SBFI/AM) is readily available commercially. Themethodology for using this probe to measure changes in cytosolic Na⁺concentration in cells other than cheek cells, and in a variety ofsituations, has been described in the scientific literature.

Proton-dependent sodium transport and changes occurring in thisparameter in association with hypertension, could also readily bemeasured by following the movement of protons (H⁺) into and out of cheekcells. Movement of H⁺ will result in changes in cheek cell cytosolic pHand this can also readily be measured by using a fluorescent probesensitive to small changes in cytosolic pH in the cheek cell. Such aprobe (BCECF/AM), together with a variety of other pH-sensitive probes,are also available commercially. Again, as for the Na⁺ sensitive probe,there is comprehensive literature related to the general use of suchfluorescent probes.

The fluorescent probe method(s) either singularly or in combinationwould greatly increase the speed at which assays could be done. Theyhave the advantage over the radioisotopic method of providing acontinuous reading of Na⁺ and/or H⁺ concentrations which would thereforeturn the assay into a kinetic one. Measurements of proton-dependentsodium uptake (or sodium-dependent proton efflux) could be done using asuitable spectrofluorimeter which is available commercially. The assaymay be designed in a manner similar to the radioisotopic method. Humancheek cells would be acidified by the methods described in thisinvention. The change in Na⁺ concentration or cell pH (H⁺concentration), following the imposition of a suitable proton gradient,would be directly measured by following the change in the fluorescentspectra of the respective-fluorescent probes(s). The above methodologycould also be applied to directly measure the intrinsic Na⁺ and H⁺permeability of the cheek cell to these ions. That is to say, that ifthe phenomenon described in this invention is explainable by anunderlying biochemical mechanism involving an intrinsic difference inthe permeability of the cheek cell membrane to these ions, then thispermeability difference could also be measured. For example, in thefinal assay it may only be necessary to determine the response of thecytosolic pH of the cheek cell to changes in the external pH; i.e. todetermine cytosolic buffering capacity in hypertensive andpre-hypertensive subjects. A fluorescent probe sensitive to cell pH suchas BCECF/AM, could also be used in this regard.

It is also quite conceivable that the fluorescent probe method couldsubsequently be simplified to a far greater extent than described above.For example, the change in cheek cell Na⁺ or pH (H⁺) concentration couldbe detected at a single time point after the mixing of the reagents wascompleted. This may only involve a single point reading using a commonlyavailable low-cost spectrofluorimeter.

Further streamlining the assay procedure described in this invention toreduce, cost, increase throughput and increase sensitivity, could easilybe done by modifying the methods used in the assay of the biochemicalmarker as previously described. One such modification could be to relyon gamma counting as opposed to liquid scintillation counting as is donein the presently described invention. Sodium-22 has the followingcharacteristics with regard to the emission of ionising radiation: Eβ,0.546 Mev; Eγ, 1.275 Mev. Gamma counting would make use of therelatively strong γ-ray emission and would negate the use ofscintillation fluid in the counting step.

Another such modification would be to separate cheek cells from theiruptake media using rapid centrifugation procedures in contrast to therapid Millipore filtration method described in the present invention.Cheek cells could be spun out of their ²² Na⁺ -containing uptakesolutions using 1.5 ml Eppendoff centfifuge tubes loaded with an aliquotof a suitable density mix of phthalates into which cheek cells wouldmigrate but the aqueous ²² Na⁺ -containing uptake solutions would not.

As mentioned previously, the kit of the invention may include anappropriate mix of organic phthalates in a suitable container. The kitmay also include a protocol for a centrifugation step to replace theMillipore filtration step in said assay procedure. Gamma counting can beused to replace beta liquid-scintillation counting.

Other Uses to which Human Cheek Epithelial (Buccal Mucosal) Cells havebeen Applied.

Although cheek cell scraping from the mouth is used extensively inteaching aspects of biology and cell structure in course work, to ourknowledge their use in more applied scientific research did not occuruntil 1984. At that time the principal investigator concerned with theinvention described herein, made use of human cheek cells to examinelipid profries in dietary and nutritional studies. These studies werereported in the following publications which have been fully listed inthe bibliography. McMurchie et at., 1983; McMurchie et at., 1984a;McMurchie et at., 1984b; McMurchie et at., 1984c; Rohan et al., 1984;Margetts et al., 1985.

Since establishing the potential for human cheek cells to offer arelatively non-invasive source of tissue, a variety of different studieson cheek cells have appeared in the literature (it is stressed however,that to our knowledge, no studies concerning hypertension in general, orion transport in particular, have appeared). These studies have beenfully listed in the bibliography and include the following: Badcock etat., 1986; Tamai et al., 1988; Lench et at., 1988; Sampugna et al., 1988and Wang et at., 1990.

In addition, the following articles have made reference to the use ofcheek cells in nutritional studies: Relation of changes in dietary fattyacid to alterations in linoleic acid content of human cheek cellphospholipids. Editorial Review, Nutrition Reviews (1984), 42 376-377;(EJM work cited in references): Methods for obtaining fat microbiopsies.Editorial comment, Nutrition Reviews (1986), 44, 200-201; (EJM workcited in references).

Testing Method 1

Materials and methods for measuring the characteristics of sodiumtransport in human cheek cells.

Collection of cheek cells

Cheek (buccal mucosal) cells were obtained in a non-invasive manner byhaving subjects swish distilled water around their mouth for a shortperiod of time, using a gentle molar scraping action. The expectoratecontaining the cheek cells was collected and a further two or three 10ml washes produced an average total yield of about 4 million cells.Samples were spun at 46,000 g×15 minutes in a Beckman J2-21 centrifugeusing a JA-20 rotor at 4° C. The packed cheek cell pellet wasresuspended in 1 ml distilled water by gentle homogenisation. Cheekcells were isolated from subjects in the morning and assayed for Na⁺ /H⁺antiporter activity on the same day.

Measurement of Na⁺ transport by the Na⁺ /H⁺ antiporter assay

Na⁺ /H⁺ antiporter activity was measured by the uptake of ²² H⁺ using amodification of the Millipore filtration technique as described bySeiler et al., (1985). All cell equilibration and transport buffers usedwere prepared by titrating a pH 5.5, buffer: 230 mM mannitol, 49 mM MES,11.2 mM N-methyl-D-glucamine (NMG), 1 mMethyleneglycol-bis-(β-amino-ethyl ether)N,N'-tetra-acetic acid (EGTA),with a pH 8.4 buffer: 230 mM mannitol, 16.8 mM glycylglycine, 26.4 mMNMG, 1 mM EGTA, to produce buffers of differing pH value as required.

The cheek cell suspension was acidified at room temperature for 3 hoursin approximately 20 volumes of pH 5.5 buffer (for proton-dependent andnon proton-dependent ²² Na⁺ uptake) or in pH 7.8 buffer (for nonproton-dependent ²² Na⁺ uptake).

Samples were centrifuged as described and resuspended in theirrespective buffers to give a final protein concentration of between 5and 15 mg/ml plus or minus the addition of 1 mM ouabain, 1 mMbumetanide, or 1 mM amiloride and further incubated at 25° C. for 15minutes.

The timed proton-dependent reaction was initiated at 25° C. by aten-fold dilution of acidified (pH 5.5) cells into uptake buffernormally at pH 7.8 which gave a final pH in the assay of 7.5 and hence atransmembrane proton gradient (inside outside) of 100:1. Measurementswere also made using uptake buffers of differing pH to produce a rangeof final proton gradients as indicated. Non proton-dependent sodiumuptake was measured by adding cells loaded at pH 5.5 or pH 7.8 to bufferof the same pH. Uptake buffer for the 100:1 proton gradient contained afinal concentration of 230 mM mannitol, 20.8 mM NMG, 18 mM MES, 10.7 mMglycylglycine, 1 mM EGTA, 1 mM Na⁺ (gluconate) and ²² Na⁺ (Cl⁻) (carrierfree) at 2×107 cpm/ml (6.4 uCi/ml), 500-1500 ug/ml cheek cell proteinplus or minus 1 mM ouabain. Na⁺ uptake was also measured in the presenceof 1 mM amiloride.

The reaction was terminated at various times as indicated by adding 25ul aliquots of the reaction mix to 4 ml ice-cold wash buffer containing1 mM NaCl, 100 mM mannitol, 100 mM MgCl₂, 8 mM HEPES, 4 mM TRIS, pH 7.2.Cells containing ²² Na⁺ were collected by rapid filtration through 0.45micron Millipore filters which were then washed twice with 4 ml ice-coldwash buffer. Filters were dried and covered with 4 mls Dupont Econofluorscintillant and counted in a Wallac 1410 beta-counter.

Measurements were made in duplicate or triplicate and background ²² Na⁺retained on filters in the absence of cheek cells was subtracted fromthe assay values. Cheek cell protein was determined by the method ofLowry et al., (1951) and controlled for the background colour readingproduced by NMG.

Rat kidney brash border membrane vesicles (BBMV) exhibiting high levelsof proton-dependent Na⁺ uptake were prepaxed according to the method ofHilden et al., (1989) using Mg²⁺ aggregation with sequentialcentrifugation. Na⁺ /H⁺ antiporter activity of BBMV was measured withevery daily set of cheek cell assays as described above as a check onthe assay procedure.

Cell counting and vital staining

Small aliquots of cells from the final pH 5.5 cell suspension werediluted in a congo red or trypan blue solution to give a final stainconcentration of 0.06% (w/v). Cells were examined under an Olympusmicroscope at ×400 magnification using an Improved Neubauerhaemocytometer. The cells were counted and inspected for their inclusionor exclusion of vital stain.

Chemicals

Amiloride HCL, bumetanide and ouabain were obtained from Sigma ChemicalCo. All other reagents were of the highest commercial grade available.

Testing Method 2

Materials and methods for measuring sodium transport in the adult cheekcell study.

Subjects

Normotensive and hypertensive subjects were recruited from a total of1100 adult male and female volunteers who participated in a bloodpressure screening program. The hypertensive group comprised subjectshaving an average of their second and third sitting diastolic bloodpressure readings of ≦95 mm Hg when measured with a Dinamap automaticblood pressure recorder and were aged between 25 and 60 years. Thecontrol group had a diastolic blood pressure of <85 mm Hg and werematched to the hypertensive group for age and gender. None of thecontrol or hypertensive subjects selected for the final study werereceiving antihypertensive medication at the time of the blood pressurescreening or during the collection of cheek cells. On the same morningof cheek cell sampling, the blood pressure was re-measured to confirmprevious readings. Characteristics of the respective study groups areshown in Table 1.

Collection of cheek cells

Cheek (buccal mucosal) cells were obtained in a non-invasive manner byhaving subjects swish distilled water around their mouth for a shortperiod of time, using a gentle molar scraping action. The expectoratecontaining the cheek cells was collected and a further two or three 10ml washes produced an average total yield of about 4 million cells.Samples were spun at 46,000 g×15 minutes in a Beckman J2-21 centrifugeusing a JA-20 rotor at 4° C. The packed cheek cell pellet wasresuspended in 1 ml distilled water by gentle homogenisation. Cheekcells were isolated from subjects in the morning and assayed for Na⁺ /H⁺antiporter activity on the same day. There was no significant differencebetween the cell yield of the normotensive group compared to thehypertensive group.

Measurement of Na⁺ uptake by the Na⁺ /H⁺ antiporter assay

Na⁺ /H⁺ antiporter activity was measured by the uptake of ²² Na⁺ using amodification of the Millipore filtration technique as described bySeiler et al.,(1985

700 ul of the cheek cell suspension was acidified at room temperaturefor 3 hours in 15 ml of Buffer I (pH 5.5) containing 230 mM mannitol, 49mM MES, 11.2 mM N-methyl-D-glucamine (NMG), and 1 mMethyleneglycol-bis-(β-amino-ethyl ether)N,N'-tetra-acetic acid (EGTA)(for proton-dependent and non proton-dependent ²² Na⁺ uptake). Theremainder (300 ul) was equilibrated in 1.5 ml of Buffer II (pH 7.8)containing 230 mM mannitol, 33.6 mM HEPES, 26.4 mM NMG and 1 mM EGTA(for non proton-dependent ²² Na⁺ uptake). Samples were centrifuged asdescribed above, and resuspended in their respective buffer plus theaddition of 1 mM ouabain, and further incubated at 25° C. for 15 mins.

The timed proton-dependent reaction was initiated at 25° C. by aten-fold dilution of pH 5.5 loaded cells (15 ul) into uptake buffer (150ul final volume) consisting of Buffer II titrated with MES to pH valuesof 7.05, 7.35, 7.65 and 7.8. This provided final proton gradients(inside→outside) of 19:1, 40:1, 75:1 and 100:1, respectively. Non protondependent ²² Na⁺ uptake was measured by adding cells loaded at pH 5.5 orpH 7.8 to uptake buffer of the same pH. For the 100:1 proton gradient,the reaction buffer contained a final concentration of 230 mM mannitol,4.9 mM MES, 30.2 mM Hepes, 24.9 mM NMG, 1 mM EGTA, 1 mM Na⁺ (gluconate)and ²² Na⁺ (Cl⁻) (carrier free) at 13.3×10⁶ cpm/ml (4.3 uCi/ml),500-1500 ug/ml cheek cell protein, plus or minus 1 mM ouabain, pH 7.5.Na⁺ uptake was also measured in the presence of 1 mM bumetanide.

The reaction was terminated at 30 seconds and 5 minutes by adding 25 ulaliquots of the reaction mix to 4 ml ice-cold wash buffer containing 1mM NaCl, 100 mM mannitol, 100 mM MgCl₂, 8 mM HEPES, 4 mM TRIS, pH 7.2.Cells containing ²² Na⁺ were collected by rapid filtration through 0.45micron Millipore filters which were then washed twice with 4 ml ice-coldwash buffer. Measurements were made in duplicate (30 seconds) andtriplicate (5 min). Filters were dried and covered with 4 ml DuPontEconofluor scintillant and counted in a Wallac 1410 beta-counter.Background values for ²² Na⁺ retained on the filters in the absence ofcheek cells were subtracted from the assay values. Cheek cell proteinwas determined by the method of Lowry et al., (1951) and controlled forthe background reading produced by NMG.

As a control for variability, weekly checks of cheek cell Na⁺ /H⁺antiporter activity of cells from a small number of normotensivevolunteers were made throughout the duration of the study. In addition,rat kidney brash border membrane vesicles exhibiting high levels ofproton-dependent sodium uptake (prepared as described by Hilden et al.,1989) using Mg²⁺ aggregation with sequential centrifugation, wereincluded with every daily set of assays as a check on the assayprocedure.

Cheek Cell counting and trypan blue staining

Small aliquots of cells were sampled from the final cell preparations atpH 5.5 and pH 7.8 and diluted ten-fold in distilled water. An aliquot ofthis dilution was added 1:1 with a neutral filtered aqueous 0.2% (w/v)trypan blue solution to give a final stain concentration of 0.1% (w/v).Cells were examined under an Olympus microscope at ×400 magnificationusing an Improved Neubauer haemacytometer that gave a viewing depth of200 um. The cells were counted and inspected for their inclusion orexclusion of trypan blue.

Chemicals

Bumetanide and ouabain were obtained from Sigma Chemical Co. Sodium-22was from New England Nuclear. All other reagents were of the highestcommercial grade available.

Testing Method 3

Materials and methods for measuring sodium transport in the adolescentcheek cell study.

Subjects

Subjects were recruited from a 3-year blood pressure tracking study.Systolic and diastolic blood pressures were measured for 504 male andfemale high school students from two schools in 1986/87 and again in1989/90. The setting up and actual running of these adolescent bloodpressure surveys were carried out by Dr PRC Howe, CSIRO, Division ofHuman Nutrition, Adelaide, to whom we are most grateful. For bothoccasions, systolic blood pressure readings were regressed on age andage squared. Weight, height and obesity were not taken into account.Deviations of each student's observation-from the expected regressionline were calculated, and from this each student was assigned a rank.The low BP and high BP tracking groups were identified from the upperand lower eight percent of the ranked sum of the two systolic bloodpressure readings. Informed consent was obtained from 35 high and 32 lowtracking adolescents. A final set of blood pressure readings in theseated position were taken using a Dinamap automatic blood pressurerecorder on the day cheek cells were collected and sodium transportassayed. Characteristics of the study group are shown in Table 3. Sixsubjects from the high BP tracking group and eight subjects from the lowBP tracking group recorded a final systolic blood pressure (on the dayof cheek cell sampling) which was less than or greater than one standarddeviation from the mean of their respective group. These subjects werenot included in the final analyses.

Collection of cheek cells

Cheek Couccal mucosal) cells were obtained in a non-invasive manner byhaving subjects swish distilled water around their mouth for a shortperiod of time, using a gentle molar scraping action. The expectoratecontaining the cheek cells was collected and a further two or three 10ml washes produced an average total yield of about 4 million cells.Samples were spun at 46,000 g×15 minutes in a Beckman J2-21 centrifugeusing a JA-20 rotor at 4° C. The packed cheek cell pellet wasresuspended by gentle homogenisation in loading buffer (pH 5.5) asdescribed below. Cheek cells were isolated from subjects in the morningand assayed for Na⁺ /H⁺ antiporter activity on the same day.

Measurement of Na⁺ transport by the Na⁺ /H⁺ antiporter assay

Na⁺ /H⁺ antiporter activity was measured by the uptake of ²² Na⁺ using amodification of the Millipore filtration technique as described bySeiler et at., (1985). All cell equilibration and transport buffers usedwere prepared by titrating a pH 5.5 buffer: 230 mM mannitol, 49 mM MES,11.2 mM N-methyl-D-glucamine (NMG), 1 mMethyleneglycol-bis-(β-amino-ethyl ether)N,N'-tetra-acetic acid (EGTA),with a pH 8.4 buffer: 230 mM mannitol, 16.8 mM glycylglycine, 26.4 mMNMG, 1 mM EGTA to produce buffers of differing pH value as requited.

Cheek cells were acidified at room temperature for 3 hours inapproximately 20 volumes of pH 5.5 loading buffer (for proton-dependentand non proton-dependent ²² Na⁺ uptake) or in pH 7.8 buffer (for nonproton-dependent ²² Na⁺ uptake). Samples were centrifuged as describedabove, and resuspended in their respective buffers to give a finalprotein concentration between 5 and 15 mg/ml plus or minus the additionof 1 mM ouabain and/or 1 mM amiloride and further incubated at 25° C.for 15 mins.

The timed proton-dependent reaction was initiated at 25° C. by aten-fold dilution of acidified (pH 5.5) cells into uptake buffer (150 ulfinal volume) normally at pH 7.8 to give a final assay pH of 7.5 andhence a transmembrane proton gradient (inside outside) of 100:1.Measurements were also made using uptake media of differing pH toproduce a range of final proton gradients as indicated. Nonproton-dependent sodium uptake was measured by adding cells loaded at pH5.5 to buffer of the same pH. Uptake buffer for the 100:1 protongradient contained a final concentration of 230 mM mannitol, 20.8 mMNMG, 18 mM MES, 10.7 mM glycylglycine, 1 mM EGTA, 1 mM Na⁺ (gluconate)and ²² Na⁺ (Cl⁺) (carrier free) at 2×10⁷ cpm/ml (6.4 μCi/ml), 500-1500ug/ml cheek cell protein, plus or minus 1 mM ouabain. Na⁺ uptake wasalso measured in the presence of 1 mM amiloride. The reaction wasterminated at various times as indicated by adding 25 ul aliquots of thereaction mix to 4 ml ice-cold wash buffer containing 1 mM NaCl, 100 mMmannitol, 100 mM MgCl₂, 8 mM HEPES, 4 mM TRIS, pH 7.2. Cells containing²² Na⁺ were collected by rapid filtration through 0.45 micron Milliporefilters which were then washed twice with 4 ml ice-cold wash buffer. Allmeasurements were made in duplicate (for 30 seconds and 10 minutes) ortriplicate (for 2 and 5 minutes). Filters were dried and covered with 4ml Dupont Econofluor scintillant and counted in a Wallac 1410beta-counter.

Background values for ²² Na⁺ retained on the filters in the absence ofcheek cells were subtracted from the assay values. Cheek cell proteinwas determined by the method of Lowry et al., (1951) and controlled forthe background colour reading produced by NMG.

As a control for variability, weekly checks of cheek cell Na⁺ /H⁺antiporter activity from a small number of normotensive volunteers weremade throughout the duration of the study. In addition, rat kidney brushborder membrane vesicles exhibiting high levels of proton-dependentsodium uptake (prepared as described by Hilden et al., 1989) using Mg²⁺aggregation with sequential centrifugation, were included with everydaily set of assays as a check on the assay procedure.

Chemicals

Amiloride HCL and ouabain were obtained from Sigma Chemical-Co.Sodium-22 was from New England Nuclear (Australia).

Satistical Method 4

All data are presented as the mean ± the standard error of the mean withthe significance of differences between means for each assaycondition/time point for the study groups, being determined by Student'sunpaired t-test.

                                      TABLE 5    __________________________________________________________________________    SUMMARY OF Na.sup.+ TRANSPORT/UPTAKE ACTIVITY IN VARIOUS TISSUES                                        Na.sup.+ UPTAKE                               ASSAY    (nmol/mg)                               TEMP  Na.sup.+ !.sub.o                                              MAXI-    AUTHOR       TISSUE/MEMBRANE                               (°C.)                                    (mM)                                        t = 1 min                                              MUM    __________________________________________________________________________    Frelin et al., 1984                 Rat heart cells                               37   3.0 5.0   20.0    Pierce et al., 1990                 Rat heart sarcolemma                               37   0.05                                        0.03  0.3    Meno et al., 1989                 Rabbit heart sarcolemma                               22   0.1 2.0   4.0    Seiler et al., 1985                 Canine heart sarcolemma                               25   1.0 13.0  15.0    Periyasamy et al., 1990                 Bovine heart sarcolemma                               22   1.0 13.0  15.0    Zadunaisky et al., 1989                 Shark retinal epithelium                               15   0.5 0.6   0.7    Ramaswamy et al., 1989                 Human ileal BBMV.sup.1                               23   1.0 5.5   5.5    Orsenigo et al, 1990                 Rat jejunum basolateral                               28   1.0: 60                                        1.0; 40                                              1.1; 40                 membrane    Moran et al., 1989                 Rat kidney cortex BBMV                               25   1.0 0.9   1.0     "           Rat kidney medulla BBMV                               25   1.0 0.4   0.5    Freiberg et al., 1982                 Rat kidney cortex BBMV                               20   1.0 0.5   5.0    Morduchowicz et al., 1989                 WKY.sup.2 kidney cortex BBMV                               21-23                                    1.0 2.1   2.3     "           SHR.sup.3 kidney cortex BBMV                               21-23                                    1.0 2.1   2.6    McMurchie    Human cheek cells    (This study) Adult-normotensive                               25   1.0       1.4    hypertensive 25            1.0      0.7                 Adolescent-low tracking                               25   1.0 2.0   2.8    high tracking                 25            1.0  1.1 1.4    __________________________________________________________________________     .sup.1 BBMVBrush border membrane vesicles     .sup.2 WKYWistar-Kyoto rat     .sup.3 SHRSpontaneously hypertensive rat

                                      TABLE 6    __________________________________________________________________________    COMPARISON OF PREVIOUS STUDIES ON CATION TRANSPORT IN RED BLOOD CELLS,    LEUCOCYTES AND PLATELETS OF HYPERTENSIVE AND NORMOTENSIVE SUBJECTS    WITH THE PRESENT CHEEK CELL MARKER FOR    HUMAN HYPERTENSION    STUDY      .sup.1 Hn                  Nn      .sup.2 Hx/Nx (%)                                  .sup.3 P                                       .sup.4 N > Hx(% H)                                                .sup.5 H < Nx (%                                                          .sup.6 OVERLAP    __________________________________________________________________________    Li.sup.+ --Na.sup.+  Countertransport-red blood cells    Weder 1986 14 31      119%    <0.05                                       --       --        --    Weinberger et al 1989               21 23 (whites)                          145%    <0.01                                       --       --        --    Weinberger et al 1989               12 11 (blacks)                          196%    <0.01                                       --       --        --    Carr et al 1990               13 23      148%    <0.01                                       3/23                                           (13%)                                                3/13                                                    (23%) 18%    Yap et al 1989               50 30      104%    N.S. --       --        --    Morgan et al 1988                9 12      371%    not given                                       --       --        --    Canessa et al 1980               36 26      229%    <0.001                                       0/36                                           (0%) 0/36                                                    (0%)   0%    Semplicini et al 1989*               41 21      133%    <0.05                                       4/21                                           (19%)                                                14/41                                                    (34%) 26%    Adragna et al 1982               22 16      176%    <0.001                                       --       --        --    Woods et al 1982               16 9       206%    <0.001                                       --       --        --    Canali et al 1981               58 46      132%    <0.001                                       --       --        --    Cusi at al 1981               45 24      130%    <0.01                                       --       --        --    Trevisan et al 1983               23 64      129%    <0.05                                       --       --        --    Williams et al 1983               54 511     123%    <0.001                                       --       --        --    Clegg et al 1982               75 39      189%    <0.001                                       --       --        --    Weder et al 1984               29 57      137%    <0.05                                       --       --        --    Wiley et al 1984               27 20      109%    N.S. --       --        --    Na.sup.+ --H.sup. +  Countertransport-Red blood cell    Morgan et al 1988                9 21      169%    <0.005                                       --       --        --    Semplicini et al 1989*               41 21      172%    <0.01                                       2/21                                           (9%) 11/41                                                    (27%) 18%    Na.sup.+ --H.sup.+  Countertransport-leucocyte    Ng et al 1989               17 17      141%    <0.05                                       0/17                                           (0%) 4/17                                                    (23%) 11.5%    Ng et al 1990               16 20      154%    <0.001                                       --       --        --    Wehling et al 1991               12 24      104%    <0.05                                       4/24                                           (17%)                                                2/12                                                    (17%) 17%    Na.sup.+ --H.sup.+  Countertransport-platelets    Livne et al 1987                7 20      195%    <0.005                                       --       --        --                                                .sup.6 N <                                                    Hx (% N)                                                          .sup.7 H > Nx (%                                                          H)    Cheek Cell proton-dependent Na.sup.+  uptake (This study)    Proton gradient (75:1)               15 19       34%    <0.0004                                       1/19                                           (5%) 1/15                                                    (7%)   6%                           Nx/Hx (%) 293%!    Slope with proton               15 19       4%     <0.003                                       2/19                                           (10%)                                                2/15                                                    (13%) 11.5%    gradient (19:1-75:1)                   Nx/Hx (%) 2400%!    __________________________________________________________________________     *The study by Semplicini et al 1989 had as its principal investigator M.     Canessa and was a follow up study of the one done by Canessa et al 1980.     .sup.1 Hn and Nn refers to the number of hypertensives (H) and     normotensives (N) in each study.     .sup.2 Hx/Nx (%) refers to the percentage increase (or decrease) between     the mean rate of cation transport in the system under investigation in     hypertensives (Hx) and normotensives (Nx).     .sup.3 Probability that differences between groups were significant as     determined by the statistical treatment used in each study. N.S., not     significant.     .sup.4 Number (%) of normotensives having a value greater than the mean o     the hypertensive group.     .sup.5 Number (%) of hypertensives having a value less than that the mean     of the normotensive group.     .sup.6 Number (%) of normotensives having a value less than that the mean     of the hypertensive group.     .sup.7 Number (%) of hypertensives having a value greater than the mean o     the normotensive group.     .sup.8 Average of (4) and (5) or (6) and (7).

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I claim:
 1. A method for determining the presence of hypertension or apre-disposition towards hypertension in a human subject, which methodcomprises:determining the sodium ion (Na⁺) uptake under an imposedproton (H⁺) concentration gradient across the membrane of isolatedsingle cheek epithelial (buccal mucosal) cells taken from said subject,wherein the rate of proton dependent sodium ion uptake by said cellsindicates the presence of hypertension or a predisposition towardshypertension in said subject.
 2. The method of claim 1, wherein theproton (H⁺) concentration gradient across the cell membrane, from theinside to the outside, is in the range of about 10:1 to about 100:1. 3.The method of claim 2, wherein the proton (H⁺) concentration gradient isabout 75:1.
 4. The method of claim 2, wherein the determination iscarried out at two or more proton concentration gradients.
 5. The methodof claim 1, wherein the cell is exposed to a medium containing a Na⁺concentration in the range of 50 micromole to 150 millimole per liter.6. The method of claim 5, wherein the Na⁺ concentration is about 1millimole per liter.
 7. The method of claim 1, wherein the determinationis carried out in the presence of an (Na⁺ -K⁺)ATPase inhibitor.
 8. Themethod of claim 7, wherein the inhibitor is ouabain.
 9. The method ofclaim 1, wherein the sodium ion uptake is determined over a periodranging from about 15 seconds to about 40 minutes during incubation atabout 25° C.
 10. The method of claim 9, wherein the sodium ion uptake isdetermined over a period of 15 seconds to 10 minutes.
 11. The method ofclaim 1, wherein the sodium ion uptake is measured by determining celluptake of radioactively labelled Na⁺.
 12. The method of claim 1, whereinNa⁺ or H⁺ are measured using an ion specific fluorescent probe.
 13. Amethod for determining the presence of hypertension or a pre-dispositiontowards hypertension in a human subject, said method comprising thesteps of:(a) establishing a proton (H⁺) concentration across themembrane of cheek epithelial (buccal mucosal) cells taken from saidsubject, the proton concentration being greater inside said cells thanoutside said cells, by performing the substeps of:(i) incubating asample of said epithelial cells taken from the subject in a first acidicmedium, (ii) contacting the acidified cells from substep (i) with asecond less acidic medium containing sodium ions (Na⁺), and (iii)separating the cells from the second less acidic medium; and (b)determining the proton-dependent uptake of sodium ions (Na⁺) by saidcells when said cells are exposed to a medium containing sodium ions(Na⁺), wherein the rate of proton dependent sodium ion uptake by saidcells indicates the presence of hypertension or a predisposition towardshypertension in said subject.
 14. The method of claim 13, wherein theproton (H⁺) concentration gradient across the cell membrane, from theinside to the outside, is in the range of about 10:1 to about 100:1. 15.The method of claim 14 wherein the proton concentration gradient isabout 75:1.
 16. The method of claim 14 wherein the determination iscarried out at two or more proton concentration gradients.
 17. Themethod of claim 13 wherein the cell is exposed to a medium containing aNa⁺ concentration in the range of 50 micromole to 150 millimole perliter.
 18. The method of claim 17 wherein the Na⁺ concentration is about1 millimole per liter.
 19. The method of claim 13 wherein thedetermination is carried out in the presence of an (Na⁺ +K⁺)ATPaseinhibitor.
 20. The method of claim 19 wherein the inhibitor is ouabain.21. The method of claim 13 wherein the Na⁺ uptake is determined over aperiod ranging from about 15 seconds to about 40 minutes duringincubation at about 25° C.
 22. The method of claim 21, wherein the Na⁺uptake is determined over a period of 15 seconds to 10 minutes.
 23. Themethod of claim 13 wherein Na⁺ uptake is measured by determining celluptake of radioactively labelled Na⁺.
 24. The method of claim 13 whereinNa⁺ H⁺ are measured using an ion specific fluorescent probe.
 25. Themethod of claim 13 when used for the determination of hypertension in anadult.
 26. The method of claim 13 when used for the determination of apredisposition to hypertension in said human subject.
 27. The method ofclaim 13, which comprises the steps of:(i) incubating a sample of saidepithelial cells taken from the human subject in the first acidicmedium, with said first acidic medium having a pH of 5.5 or higher; (ii)contacting the so acidified cells with the second less acidic mediumcontaining said sodium ions (Na⁺), with said second acidic medium havinga pH of 7.8 or lower; (iii) separating the cells from the second lessacidic medium; and (iv) determining the uptake of said sodium ions (Na⁺)by the cells.
 28. An assay kit which is useful in a method fordetermining the presence of hypertension or a predisposition tohypertension in a human subject, the kit comprising:a first acidicmedium having a pH of 5.5 or higher in a suitable container foracidifying epithelial cells isolated from a human subject; a second lessacidic medium having a pH of 7.8 or lower containing sodium ions (Na⁺)in a suitable container; a third medium containing a final sodium ion(Na⁺) concentration therein of from 50 micromoles to 150 millimoles perliter in a suitable container; an indicator for the detection of sodiumion (Na⁺) in a suitable container; and an inhibitor of (Na⁺ +K⁺) ATPaseactivity in a suitable container.
 29. The assay kit according to claim28, wherein said epithelial cells are isolated single cheek epithelial(buccal mucosal) cells.
 30. The assay kit according to claim 28, whereinthe final sodium ion (Na⁺) concentration in said third medium is about 1millimole per liter.
 31. The assay kit according to claim 28, whereinthe indicator for sodium ion (Na⁺) concentration is an ion specificfluorescent probe specific for Na⁺, or H⁺, or Na⁺ and H⁺.
 32. The assaykit according to claim 28, wherein the inhibitor of (Na⁺ +K⁺) ATPaseactivity is ouabain.
 33. The assay kit according to claim 28, furthercomprising a predetermined mix of organic phthalates in a suitablecontainer.
 34. The assay kit according to claim 28, further comprisingradioactive sodium ion (Na⁺) in a suitable container.
 35. The assay kitaccording to claim 29, wherein the final sodium ion (Na⁺) concentrationin said third medium is about 1 millimole per liter.
 36. The assay kitaccording to claim 29, wherein the indicator for sodium ion (Na⁺)concentration is an ion specific fluorescent probe specific for Na⁺, orH⁺, or Na⁺ and H⁺.
 37. The assay kit according to claim 29, wherein theinhibitor of (Na⁺ +K⁺) ATPase activity is ouabain.
 38. The assay kitaccording to claim 29, further comprising a predetermined mix of organicphthalates in a suitable container.
 39. The assay kit according to claim29, further comprising radioactive sodium ion (Na⁺) in a suitablecontainer.